干旱胁迫下割手密根系转录组差异表达分析

doi:10.3864/j.issn.0578-1752.2017.06.017

摘要/Abstract

摘要： 【目的】探明云南割手密82-114受干旱胁迫的内在分子机制，挖掘与抗旱密切相关的功能基因，提高割手密抗旱基因的研究与利用。【方法】以干旱胁迫24、48和72 h的云南割手密82-114的根系进行Illumina HiSeqTM 4000高通量转录组测序分析，将组装得到的Unigene分别与Swiss-Prot、Nr、KOG、Pfam和KEGG数据库进行比对，利用RPKM来衡量各样本间的基因表达丰度，以FDR≤0.05和|log2 fold change|≥1来评估样本间的差异表达基因，通过Gene Ontology（GO）数据库、KEGG pathway数据库对不同干旱胁迫样本差异表达基因的功能和参与的调控路径进行分析。【结果】未胁迫处理的CK与胁迫24、48和72 h后的样本转录组分别检测到134 724、130 368、133 564和131 321个表达基因，与CK相比，各胁迫样本显著上调（或下调）的差异表达基因分别有3 061（1 302）、2 304（2 841）和3 236（2 525）个；以FDR值= 0为筛选条件，3个样本的极显著差异表达基因主要参与转录激活、水分运输、DNA结合、ATP结合及细胞膜、跨膜运输和防御反应等有关代谢活动。GO富集分析表明：3个胁迫处理的样本在生物学途径中富集最多的类别并不完全相同，其中，胁迫24 h与48 h的样本富集最多的类别是与DNA依赖型转录和转录调节子相关的基因，胁迫72 h样本富集最多的类别是与翻译相关的基因；而在细胞组件和分子功能中，3个胁迫样本富集最多的基因类别均是与内在的膜、核和ATP结合相关的基因。KEGG富集分析表明：胁迫24 h的样本有2 248个差异表达基因参与了107条代谢途径，胁迫48 h的样本有2 114个差异表达基因参与了130条代谢途径，胁迫72 h的样本有2 392个差异表达基因参与了144条代谢途径；经KEGG显著性富集分析，筛选到鞘糖脂生物合成途径、MAP激酶信号途径和ABC转运蛋白等与非生物胁迫或逆境相关。【结论】获得3个干旱胁迫样本与CK样本间差异表达基因的变化及其功能信息，发现参与云南割手密82-114干旱胁迫响应相关的细胞外信号调节激酶、磷脂酶D、β-氨基己糖苷酶和ATP结合盒B亚族（MDR/TAP）-1等，获得在整个干旱胁迫时期稳定上调表达基因70个，稳定下调表达基因11个，通过同源基因序列的比对分析，阐明这些基因在各自的代谢通路中被强烈诱导或抑制表达，显示与干旱胁迫存在密切关系。

关键词: 割手密, 干旱, 转录组, 差异表达基因

Abstract: 【Objective】The objective of this study is to understand the inner molecular mechanisms of Saccharum spontaneumclone named Yunnan 82-114 in response to water stress, and mine these genes closely related to drought tolerance, improve the utilization efficiency of Saccharum spontaneum in sugarcane drought-resistant breeding program. 【Method】llumina HiSeqTM 4000, a high-through transcriptome sequencing technology, was applied to obtain the transcriptome differential expression data of Yunnan82-114 roots under drought stress treatment for 24h, 48h, and 72h. These assembled unigenes above were compared with database of Swiss-Prot, Nr, KOG, Pfam, and KEGG, respectively, then the abundance of gene expression among different samples were screened according to transcriptome data by using RPKM method, and the differentially expressed genes among the treated samples were estimated by referring to the standard of FDR≤0.05 & |log2 fold change|≥1. Function and pathway of those different expression genes were also investigated using Gene Ontology（GO）database and KEGG pathway database.【Result】Total 134 724, 130 368, 133 564, and 131 321 expressing genes were got, respectively, from the untreated control and three treated samples(24 h, 48 h and 72 h). Compared the control with the treated samples, about 3 061 (1 302), 2 304 (2 841) and 3 236 (2525) genes whose expression was significantly up-regulated (or down-regulated) were detected, respectively. When the FDR value was set to be 0 as a selection criterion, some genes performing extreme significantly different expression between control and treated samples were acquired, they mainly involved in transcriptional activation, water transport, DNA binding, ATP binding, membrane/ transmembrane transport, and defense response. GO enrichment analysis showed that some differences in the biological approach categories existed among the treated samples, for the samples treated for 24 h and 48h, the most enriched category was DNA-dependent transcription and transcription factor, then to the samples treated for 72 h, the enriched category was the translation-related genes. At the aspects of cellular components and molecular functions, the enriched category was inner membrane, nucleus, and ATP binding related genes in the three treated samples. KEGG enrichment analysis illustrated that 2 248 differentially expressed genes involved in 107 pathways in 24 h-treated sample, 2 114 in 130 in 48h-treated sample, and 2 392 in 144 in 72 h-treated sample. Finally, some genes related to abiotic stress and resistance were screened via KEGG significance enrichment analysis, which include glycosylsphingolipid biosynthesis, MAP kinase signal transduction, and ABC translocator.【Conclusion】Expression pattern and function information of differentially expressed genes between three water-stressed samples and a control were analyzed. Some genes like extracellular signal-regulated kinase, phospholipase D, hexosaminidase, and ATP-binding cassette B-1 were found to involve in response to water stress for Yunnan82-114. About 70 significant up-regulated expression genes at transcription level were obtained, but there were 11 genes exhibiting down-regulated expression, this implied that these genes induced or suppressed fiercely by drought stress have obvious relationships with drought tolerance of Yunnan82-114.

Key words: Saccharum spontaneum, drought, transcriptomes, differentially expressed genes

刘洪博，刘新龙，苏火生，陆鑫，徐超华，毛钧，林秀琴，李纯佳，李旭娟，字秋艳. 干旱胁迫下割手密根系转录组差异表达分析[J]. 中国农业科学, 2017, 50(6): 1167-1178.

LIU HongBo, LIU XinLong, SU HuoSheng, LU Xin, XU ChaoHua, MAO Jun, LIN XiuQin, LI ChunJia, LI XuJuan, ZI QiuYan. Transcriptome Difference Analysis of Saccharum spontaneum Roots in Response to Drought Stress[J]. Scientia Agricultura Sinica, 2017, 50(6): 1167-1178.

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